This continuation application from Dr. Michael Young describes a series of experiments to investigate a system of developmental signaling in Drosophila melanogaster. A critical component of the pathway under study is the Notch transmembrane receptor. Notch and several other "neurogenic " genes control cell fate choice during differentiation within all germ layers. Notch loss-of-function mutations typically overproduce neuroblasts and muscle founder cells at the expense of epidermis and other mesodermal cells respectively. Notch is a part of a conserved protein family with other members having been identified in C. elegans, Xenopus, mouse and man. All Notch-like molecules encode transmembrane proteins with large extracellular domains consisting primarily of tandemly repeated EGF-like sequences (36 for Notch) and smaller cytoplasmic domains containing cdc10/ankyrin repeats. In Drosophila and C. elegans mutations in different EGF repeats generate distinct mutant phenotypes, but so far only two ligands have been identified and they interact with adjacent EGF modules. On the intracellular side, the cdc10/ankyrin repeats are thought to modulate protein-protein interactions. In particular, these repeats are required for an interaction with the Suppressor of Hairless {Su(H)} transcription factor and may mediate Su(H) transcriptional control of target genes contained in the enhancer of split complex. The mechanism of how Notch activates Su(H) function is not known, but the speculation is that a ligand dependent processing event takes place which liberates the cytoplasmic portion of Notch allowing it to localize with Su(H) in the nucleus. The identities of all the ligands that can stimulate the putative processing event as well as the factors responsible for the mediating the cleavage are presently undefined. In addition, the extent to which other intracellular proteins beside Su(H) may help transduce the Notch signal has not been fully explored. Dr. Young's research plan outlines five specific aims that focus on elucidating the mechanism of Notch signal transduction. 1) He will determine whether Notch activation in wildtype flies involves production of a nuclear Notch signal. 2) A reporter assay has been generated in cultured Drosophila cells that involves Notch activation of the transcription factor Suppressor of Hairless. The system will be used to determine how activated forms of Notch produce transcription signals and to test for direct functional involvement of novel receptor binding proteins. 3) Anti-neurogenic Notch genes will be used in genetic screens for modifiers, among which is speculated to be an intracellular Notch protease. 4) Proteins directly interacting with the intracellular domain of Notch will be sought using modified yeast two hybrid screens. 5) Several new Notch extracellular binding proteins that were isolate in an f1 phage biopanning screen will be tested for functional activity in vivo.